Most genetic differences between individuals are single-nucleotide polymorphisms (SNPs). At most SNP positions, there are two possible alleles. Such SNPs are distributed throughout the genome at frequency of about 1 per 1,000 base pairs. Several hundred thousand SNP markers are now available in public databases. These databases should facilitate the identification of genetic markers associated with disease. SNP scoring assays can determine which allele an individual has for an SNP of interest. A series of suitably designed SNP scoring assays can be used to link an SNP with a disease. However, in order to associate thousands of SNP markers with individual diseases, millions of SNP scoring assays must be carried out in large populations. Therefore, efficient methods to rapidly score SNPs at low cost for large populations are needed to utilize genetic markers for the mapping of disease genes and the effective diagnosis, treatment or prevention of the disease.
Several methods based on the migration of Holliday junctions in nucleic acids are capable of detecting SNPs under certain conditions (see, e.g., U.S. Pat. No. 6,013,439, U.S. Pat. No. 6,232,104 B1 and PCT publication WO 01/69200). In general, these methods can be used to detect differences between the sequences of two nucleic acids. For example, these methods can be used to detect sequence differences between a first nucleic acid with a known sequence and a second nucleic acid with an unknown sequence. For these methods to be used accurately, the two nucleic acids should have the same sequences everywhere except at the site of the polymorphism. If the nucleic acids have the same genotype, Holliday junction migration can proceed through the lengths of the nucleic acids. If the sequences of the nucleic acids differ, Holliday junction migration can halt at a site of sequence difference under the appropriate conditions. Detection of stabilized Holliday junctions can indicate that the sequence of the second nucleic acid differs from that of the first nucleic acid.
Although current Holliday junction detection methods can be used to detect differences between the sequences of two nucleic acids, they cannot be applied to compare the genotypes of all nucleic acids accurately. Current Holliday junction methods require that the nucleic acids have a minimum of about 100 base pairs, especially for the detection of single base mismatches. Most single mismatches between two nucleic acids shorter than 100 base pairs in length do not create sufficient energy barriers to impede branch migration for detection of stable Holliday junctions. Thus, most single base mismatches, for example at the site of an SNP, are capable of detectably impeding Holliday junction migration only if the nucleic acids are at least about 100 base pairs in length. Current methods are therefore often limited to the determination of the genotypes of nucleic acids at least about 100 base pairs in length.
Yet even when applied to nucleic acids greater than 100 base pairs in length, current methods can suffer from inaccuracies. Frequently, more than one SNP can be found within nucleic acid, e.g., a sample of genomic DNA, that is 100 base pairs in length. The frequency only increases as the length of the nucleic acid increases. If a pair of nucleic acids has more than one SNP in their lengths, current Holliday junction methods can only indicate that the nucleic acids differ in genotype somewhere in their sequences. They cannot resolve the difference or differences to particular SNPs.
There is a need for improved Holliday junction methods of scoring SNPs that can be used to accurately identify the genotype of an individual SNP in a given nucleic acid. The methods should be capable of application to nucleic acids of any length, including those of less than 100 base pairs. They should also be capable of resolving the genotypes of individual SNPs, even in nucleic acids comprising multiple SNPs.